LOCUS       sequence-263527-        7069 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Live dual labeling of the actin network and polymerizing 
            microtubules for dynamic analysis of the cytoskeleton.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7069)
  AUTHORS   Dupraz S, Hilton BJ, Husch A, Santos TE, Coles CH, Stern S, 
            Brakebusch C, Bradke F
  TITLE     RhoA Controls Axon Extension Independent of Specification in the 
            Developing Brain.
  JOURNAL   Curr Biol. 2019 Nov 18;29(22):3874-3886.e9. doi: 
            10.1016/j.cub.2019.09.040. Epub 2019 Oct 31.
  PUBMED    31679934
REFERENCE   2  (bases 1 to 7069)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cub.2019.09.040"; journalName: "Curr Biol"; date: 
            "2019-11-18- 18"; volume: "29"; issue: "22"; pages: "3874-3886.e9"
COMMENT     Sequence Label: pCMV-EB3mCherry_cmv-LifeActGFP
FEATURES             Location/Qualifiers
     source          1..7069
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     regulatory      282..291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1140..1149
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1146..1856
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1291..1309)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1559..1578
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1979..2100
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2244..2265)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     enhancer        2319..2622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2623..2826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             2893..2943
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      2959..2968
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2965..2967,2968..2970,2971..3684)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2965..2967,2968..2970,2971..3681)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3010..3031)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3271..3290)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3618..3639
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3714..3733)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3779..3834
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3833..3852)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4195..4211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4195..4211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4208..4230)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4219..4235
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4243..4249,4250..4267,4268..4273))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4288..4309
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4368..4563
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4414..4549
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4569..4703
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4771..4788)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4942..5530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5022..5041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5701..6492,6493..6561))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6324..6343
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6562..6666)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6697..7069
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgagctcaag cttcgaattc tgcagtcgac tgccaccatg gccgtcaatg
      301 tgtactccac atctgtgacc agtgaaaatc tgagtcgcca tgatatgctt gcatgggtca
      361 acgactccct gcacctcaac tataccaaga tagaacagct ttgttcaggg gcagcctact
      421 gccagttcat ggacatgctc ttccccggct gtgtgcactt gaggaaagtg aagttccagg
      481 ccaaactaga gcatgaatac atccacaact tcaaggtgct gcaagcagct ttcaagaaga
      541 tgggtgttga caaaatcatt cctgtagaga aattagtgaa aggaaaattc caagataatt
      601 ttgagtttat tcagtggttt aagaaattct ttgacgcaaa ctatgatgga aaggattaca
      661 accctctgct ggcgcggcag ggccaggacg tagcgccacc tcctaaccca ggtgatcaga
      721 tcttcaacaa atccaagaaa ctcattggca cagcagttcc acagaggacg tcccccacag
      781 gcccaaaaaa catgcagacc tctggccggc tgagcaatgt ggcccccccc tgcattctcc
      841 ggaagaatcc tccatcagcc cgaaatggcg gccatgagac tgatgcccaa attcttgaac
      901 tcaaccaaca gctggtggac ttgaagctga cagtggatgg gctggagaag gaacgtgact
      961 tctacttcag caaacttcgt gacatcgagc tcatctgcca ggagcatgaa agtgaaaaca
     1021 gccctgttat ctcaggcatc attggcatcc tctatgccac agaggaagga ttcgcacccc
     1081 ctgaggacga tgagattgaa gagcatcaac aagaagacca ggacgagtac ctggatcccg
     1141 ccaccatggt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct
     1201 tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg
     1261 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     1321 tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga
     1381 agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg
     1441 agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctgc
     1501 aggacggcga gttcatctac aaggtgaagc tgcgcggcac caacttcccc tccgacggcc
     1561 ccgtaatgca gaagaagacc atgggctggg aggcctcctc cgagcggatg taccccgagg
     1621 acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
     1681 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca
     1741 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1801 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtaaagcg
     1861 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     1921 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     1981 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2041 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     2101 aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     2161 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2221 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactagta gtaatcaatt
     2281 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     2341 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     2401 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     2461 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     2521 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     2581 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     2641 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     2701 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     2761 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     2821 agagctggtt tagtgaaccg tcagatccgc tagcgctacc ggactcagat ctcgagctca
     2881 agcttcgaat tcatgggtgt cgcagatttg atcaagaaat tcgaaagcat ctcaaaggaa
     2941 gaaggggatc caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     3001 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     3061 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     3121 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     3181 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3241 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3301 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3361 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3421 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3481 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3541 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3601 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3661 ggcatggacg agctgtacaa gtaaagcggc ctggctcaca aataccactg agatcttttt
     3721 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa
     3781 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     3841 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg
     3901 gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag
     3961 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt
     4021 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc
     4081 ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc
     4141 cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata
     4201 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4261 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4321 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag
     4381 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4441 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4501 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4561 aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4621 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4681 atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg
     4741 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4801 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4861 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4921 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4981 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5041 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5101 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5161 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5221 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5281 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5341 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5401 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5461 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5521 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5581 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5641 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5701 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5761 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5821 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5881 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5941 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6001 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6061 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6121 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6181 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6241 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6301 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6361 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6421 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6481 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6541 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6601 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6661 tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg actagtagta
     6721 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     6781 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     6841 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     6901 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     6961 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     7021 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatg
//
